Preparations and methods for the treatment of t cell mediated diseases

ABSTRACT

Metabolizable lipid emulsions, such as Intralipid and Lipofundin, are excellent vehicles for peptide therapy of autoimmune diseases and of other TH1 T cell mediated diseases or conditions, as it promotes a TH1 to TH2 cytokine shift. Such emulsions may be used in conjunction with an antigen recognized by inflammatory T cells associated with the pathogenesis of a T cell mediated disease or condition for the therapeutic treatment of such

FIELD OF THE INVENTION

[0001] The present invention relates to vaccine therapy for T-cellmediated diseases, and in particular to therapeutic preparationscomprising antigens recognized by T cells involved in the pathogenesisof T cell mediated diseases, such as autoimmune diseases, and ametabolizable lipid emulsion as a biologically active carrier.

BACKGROUND OF THE INVENTION

[0002] Autoimmune disorders, e.g., insulin-dependent diabetes mellitus(IDDM or type I diabetes), multiple schlerosis, rheumatoid arthritis andthyroiditis, are characterized by reactivity of the immune system to anendogenous antigen, with consequent injury to tissues. These immuneresponses to self-antigens are maintained by the persistent activationof self-reactive T lymphocytes.

[0003] T cells of the CD4 “helper” type have been divided into twogroups by the characteristic cytokines they secrete when activated(Mosmann and Coffman, 1989). TH1 cells secrete IL-2, which induces Tcell proliferation, and cytokines such as IFN-γ, which mediate tissueinflammation. TH2 cells, in contrast, secrete IL-4 and IL-10. IL-4 helpsT cells secrete antibodies of certain IgG isotypes and suppresses theproduction of TH1 inflammatory cytokines (Banchereau et al., 1994).IL-10 indirectly inhibits TH1 activation by affectingantigen-presentation and inflammatory cytokine production by macrophages(Moore et al., 1993). It is the TH1 cells which contribute to thepathogenesis of organ-specific autoimmune diseases. TH1-type responsesalso appear to be involved in other T cell mediated diseases orconditions, such as contact dermatitis (Romagnani, 1994).

[0004] Peptides suitable for immunologically specific therapy of anautoimmune disease are peptides that are recognized by T cells involvedin the pathogenesis of the autoimmune disease. Each autoimmune diseasewill have its ideal peptide for use in therapy. A disease like multiplesclerosis involving T cells reactive to self-antigens such as myelinbasic protein (MBP) (Allegreta et al., 1990) will require a peptide ofmyelin basic protein for its therapy, as for example those described byOta et al., 1990.

[0005] The present inventors have shown that autoimmune diseases such astype I diabetes mellitus may be treated by administering a suitablepeptide in an oil vehicle. NOD mice spontaneously develop type Idiabetes caused by autoimmune T cells that attack the insulin-producingβ cells of the islets. The autoimmune attack is associated with T-cellreactivity to a variety of self-antigens including a peptide of the 60kDa heat shock protein (hsp 60) and peptides of glutamic aciddecarboxylase (GAD). Thus, for example, spontaneous diabetes developingin the NOD/Lt strain of mice could be treated with a peptide designatedp277 corresponding to positions 437-460 of the human hsp 60 sequence(PCT Patent Publication No. WO90/10449; D. Elias and I. R. Cohen,Peptide therapy for diabetes in NOD mice, The Lancet 343:704-06, 1994);with variants of the p277 peptide in which one or both cysteine residuesat positions 6 and/or 11 have been replaced by valine and/or the Thrresidue at position 16 is replaced by Lys (see PCT PublicationWO96/19236) and with peptides designated p12 and p32 corresponding topositions 166-185 and 466-485, respectively, of the human hsp60sequence. See Israel Patent Application No. 114,407 of the sameapplicant of the present application, filed on Jun. 30, 1995. See alsoPCT application No. PCT/US96 . . . , filed Jul. 1, 1996, claimingpriority from said Israel application no. 114,407, the entire contentsof which are hereby incorporated by reference.

[0006] Peptide therapy for treatment of IDDM using p12, p32, p277 orvariants thereof, was found by the present inventors to be effectivewhen the peptide was administered to mice subcutaneously (sc) in an oilvehicle such as an emulsion of mineral oil known as incomplete Freund'sadjuvant (IFA). However, IFA as well as complete Freund's adjuvant (CFA;a preparation of mineral oil containing various amounts of killedorganisms of Mycobacterium) are not allowed for human use because themineral oil is not metabolizable and cannot be degraded in the body.Therefore, it would be desirable to discover an effective vehicle forpeptide therapy that would be metabolizable.

[0007] Several fat emulsions have been in use for many years forintravenous nutrition of human patients. Two of the available commercialfat emulsions, known as Intralipid (“Intralipid” is a registered trademark of Kabi Pharmacia, Sweden, for a fat emulsion for intravenousnutrition, described in U.S. Pat. No. 3,169,094) and Lipofundin (aregistered trade mark of B. Braun Melsungen, Germany) contain soybeanoil as fat (100 or 200 g in 1,000 ml distilled water: 10% or 20%,respectively). Egg-yolk phospholipids are used as emulsifiers inIntralipid (12 g/l distilled water) and egg-yolk lecithin in Lipofundin(12 g/l distilled water). Isotonicity results from the addition ofglycerol (25 g/l) both in Intralipid and in Lipofundin. These fatemulsions are quite stable and have been used for intravenous nutritionof patients suffering from gastrointestinal or neurological disorders,which prevent them from receiving nutrition orally, and thus theyreceive the calories needed to sustain life. Usual daily doses are of upto 1 liter daily.

[0008] U.S. Pat. No. 4,073,943 issued on Feb. 14, 1978 to Wretlind etal. and U.S. Pat. No. Re. 32,393 issued on May 29, 1990 as reissuepatent of U.S. Pat. No. 4,168,308 issued on Sep. 18, 1979 to Wretlind etal., describe a carrier system for use in enhancing parenteral,particularly intravenous, administration of a pharmacologically active,oil-soluble agent, comprising a stable, oil-in-water emulsion containinga pharmacologically inert lipoid as a hydrophobic phase dispersed in ahydrophilic phase, said lipid being dispersed in the emulsion as finelydivided particles having a mean particle size less than 1 micron toachieve rapid onset of an acceptable therapeutic effect, said carriersystem being used with an effective dose of said pharmacologicallyactive, oil-soluble agent predominantly dissolved in said lipoid at afraction ratio thereto in the hydrophobic phase, said therapeutic effectbeing attributable to said effective dose of the active agent. Thiscarrier system is said to be suitable for administration of awater-insoluble or water-soluble, oil-soluble pharmacologically activeagent that is predominantly dissolved in the lipoid phase. Examples ofsuch pharmacologically active agents are depressants, anaesthetics,analgesics, stimulants, spasmolytics, muscle relaxants, vasodepressantsand diagnostic, e.g. X-ray contrast, agents. The carrier system is saidto enhance the diagnostic or therapeutic effect of the agent with arapid onset accompanied by a reduced incidence of injury to bodytissues.

[0009] Intralipid has been proposed as a non-irritating vehicle forseveral adjuvants for use in vaccines such as, for example,6-O-(2-tetradecylhexadecanoyl)- and6-O-(3-hydroxy-2-docosylhexacosanoyl)-N-acetylmuramyl-L-alanyl-D-isoglutamine(Tsujimoto et al., 1986 and 1989), avridine (Woodard and Jasman, 1985),N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl) propanediamine (CP-20,961)(German Patent Application No. DE 2945788; Anderson and Reynolds, 1979;Niblack et al., 1979). Kristiansen and Sparrman, 1983, have disclosedthat the immunogenicity of hemagglutinin and neuraminidase in mice ismarkedly increased after adsorption onto lipid particles constitutingIntralipid.

[0010] None of the above publications describe the use of Intralipid asa vehicle for peptides in the treatment of autoimmune diseases, nor hasthere been any disclosure that Intralipid could mediate a shift of theimmune response from a TH1-type response to a TH2-type response.

SUMMARY OF THE INVENTION

[0011] It has now been found, in accordance with the present invention,that metabolizable lipid emulsions, such as Intralipid and Lipofundin,can act as vehicles for peptide therapy of autoimmune diseases and ofother TH1 T cell mediated diseases or conditions. It has been furtherfound that this activity is associated with a TH1 to TH2 cytokine shift.

[0012] The present invention thus relates to a therapeutic preparationfor the treatment of an autoimmune disease or other T cell mediateddisease or condition, comprising a peptide or other antigen and abiologically active lipid carrier, wherein the peptide or other antigenis one recognized by inflammatory T-cells associated with thepathogenesis of said disease or condition, and wherein the biologicallyactive lipid carrier is a fat emulsion comprising 10-20% triglyceridesof plant and/or animal origin, 1.2-2.4% phospholipids of plant and/oranimal origin, 2.25-4.5% osmo-regulator, 0-0.05% anti-oxidant, andsterile water to 100%.

[0013] The triglycerides and phospholipids of plant or animal origin mayderive from any suitable vegetable oil, such as soybean oil, cottonseedoil, coconut oil or olive oil, or from egg-yolk or bovine serum.Preferably, the triglycerides are derived from soybean oil and thephospholipids are derived from soybean or from egg-yolk. Preferably, thetriglycerides/phospholipids weight ratio is about 8:1.

[0014] Any suitable osmo-regulator may be added to the fat emulsion,preferably glycerol, xylitol or sorbitol. The fat emulsion mayoptionally comprise an anti-oxidant, for example 0.05% tocopherol.

[0015] In one embodiment of the invention, the fat emulsion as definedabove is processed by centrifugation, e.g. at 10,000 g or higher, thusforming a small triglyceride-rich (about 90% triglycerides) layer on thetop of a phospholipid-enriched aqueous dispersion containing about 1:1triglycerides:phospholipids, and this latter aqueous dispersion is usedas the lipid vehicle in the preparations of the invention.

[0016] In one preferred embodiment of the invention, the preparation isfor the treatment of insulin-dependent diabetes mellitus (IDDM) andcomprises a peptide derived from the human heat shock protein 60 (hsp60)that is recognized by inflammatory T-cells associated with thepathogenesis of IDDM, wherein said peptide is selected from the group ofpeptides appearing in the following Table 1: TABLE 1 Amino acid sequencePeptides Sequence ID No: (one letter code) p3 1 (31-50)KFGADARALMLQGVDLLADA p10 1 (136-155) NPVEIRRGVMLAVDAVIAEL p11 1(151-170) VIAELKKQSKPVTTPEEIAQ p12 1 (166-185) EEIAQVATISANGDKEIGNI P141 (195-214) RKGVITVXDGKTLNDELEII p18 1 (255-274) QSIVPALEIANAHRKPLVIIAp20 1 (286-305) LVLNRLKVGLQVVAVKAPGF p24 1 (346-365)GEVIVTKDDAMLLKGKGDKA p29 1 (421-440) VTDALNATRAAVEEGIVLGG p30 1(436-455) IVLGGGCALLRCIPALDSLT p32 1 (466-485) EIIKRTLKIPAMTIAXNAGV p351 (511-530) VNMVEKGIIDPTKVVRTALL p39 1 (343-366) GKVGEVIVTKDDAM p277 1(437-460) VLGGGCALLRCIPALDSLTPANED p277 (Val⁶⁾ *2VLGGGVALLRCIPALDSLTPANED p277 (Val¹¹⁾ **3 VLGGGCALLRVIPALDSLTPANED p277(Val⁶-Val¹¹) ***4 VLGGGVALLRVIPALDSLTPANED

[0017] The invention further relates to a method for therapy of asubject suffering from an autoimmune disease or other TH1 mediateddisease or condition, which comprises administering to said subject aneffective amount of a therapeutic preparation according to theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows anti-p277 antibody production in NOD mice treatedwith the peptide p277(Val⁶-Val¹¹) in: (i) Intralipid or (ii)phosphate-buffered saline (PBS), as described in Example 2.

[0019]FIG. 2 shows TH2-dependent antibody isotypes induced in NOD miceby treatment with the peptide p277(Val⁶ -Val¹¹) in Intralipid, asdescribed in Example 3.

[0020] FIGS. 3A-B show that p277(Val⁶-Val¹¹)/Intralipid therapy inducesin NOD mice a specific switch in the profile of cytokines produced bythe T-cells reactive to the p277(Val⁶-Val¹¹) peptide, as described inExample 4. FIG. 3A shows that there is a reduction of TH1 (IL-2, IFN-γ)and elevation of TH2 (IL-4, IL-10) cytokines after treatment of the micewith the p277(Val⁶-Val¹¹) peptide in Intralipid and incubation of thespleen cells with p277(Val⁶-Val¹¹); FIG. 3B shows that there is nochange in the cytokines after treatment of the mice with thep277(Val⁶-Val¹¹) peptide in Intralipid and incubation of the spleencells with Con A.

[0021]FIG. 4 shows that spontaneous T-cell proliferative responses top277(Val⁶-Val¹¹) is reduced after treatment with the p277(Val⁶-Val¹¹)peptide in Intralipid, as described in Example 5.

[0022]FIG. 5 shows that treatment of rats with myelin basic proteinpeptide p71-90 in Intralipid reduces the severity of experimentalautoimmune encephalomyelitis (EAE), as described in Example 6.

[0023]FIG. 6 shows that treatment of rats with myelin basic proteinpeptide p71-90 in IFA reduces the severity of experimental autoimmuneencephalomyelitis (EAE), as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

[0024] According to the present invention, it was found thatp277(Val⁶-Val¹¹)-peptide treatment, in an appropriate carrier,down-regulated the spontaneous T-cell proliferative responses toepitopes of both hsp60 and GAD and abolished the production ofautoantibodies to hsp60, to GAD and to insulin. Arrest of the diseaseprocess was associated, not with T-cell tolerance or energy, but with ashift in the cytokines produced by the autoimmune T cells reactive top277(Val⁶-Val¹¹) from a TH1-like profile (IL-2, IFNγ) to a TH2-likeprofile (IL-4, IL-10). The modulation was immunologically specific; thespontaneous T-cell response of the treated mice to a bacterial hsp60peptide remained in the TH1 mode. Thus, the diabetogenic processcharacterized by autoimmunity to several self antigens can be curedusing one of the antigens, e.g., peptide p277(Val⁶-Val¹¹).

[0025] The association of p277(Val⁶-Val¹¹) therapy with a switch inreactivity to p277(Val⁶-Val¹¹) from T-cell proliferation to antibodiesindicates that the therapeutic effect results from a shift in thepredominant cytokines produced by the autoimmune T cells in the treatedmice. TH1 cells secrete IL-2, which induces T-cell proliferation, andcytokines such as IFN-γ, which mediate tissue inflammation, therebycontributing to the pathogenesis of the disease; TH2 cells, in contrast,secrete IL-4 and IL-10. IL-4 helps B cells secrete antibodies of certainIgG isotypes and suppresses the production of TH1 inflammatorycytokines. IL-10 indirectly inhibits TH1 activation by affectingantigen-presentation and inflammatory cytokine production bymacrophages. Thus, TH2 cells suppress TH1 activity (see Liblau et al.,1995). The shift from TH1 to TH2-like behavior was supported by analysisof the isotypes of the antibodies produced before and afterp277(Val⁶-Val¹¹) therapy.

[0026] The fact that the mechanism of the therapeutic effect of thepeptide in a lipid vehicle treatment is shown to involve a TH1→TH2cytokine shift, provides the possibility of using the TH1→TH2 shift asevidence that the treatment was effective and did induce a beneficialresponse. In other words, the TH1→TH2 shift can serve as a surrogatemarker of the response to treatment. For example, the lack of the shiftcan indicate a need for a second treatment. See Israel PatentApplication No. 114,459 filed on Jul. 5, 1995, and the corresponding PCTapplication filed on even date herewith, the entire contents of whichare hereby incorporated herein by reference.

[0027] The lipid emulsions of the present invention, when used as avaccine adjuvant with the antigenic substance to which the T cellsinvolved in the disease or condition being treated are active, serve tomediate a shift from a TH1 T cell response prior to treatment to a TH2 Tcell response after treatment. This finding establishes that such lipidemulsions are tolerogenic biologically active carriers which can be usedin vaccines for the treatment of any TH1 mediated disease or condition.In such vaccines, the antigen provides the immunological specificity fora therapeutic effect while the biologically active carrier of thepresent invention provides the biological outcome, i.e., the TH1→TH2shift. Because of the shift mediated by said biologically active carrierof the present invention, diseases with a spectrum of autoreactivitiescan be turned off with a single antigen/carrier combination capable ofinducing a T cell cytokine shift.

[0028] A preferred use in accordance with the present invention is inthe treatment of organ-specific autoimmune diseases which are mediatedby TH1 cells. Such diseases include, but are not limited to, autoimmunediseases such as IDDM, rheumatoid arthritis, multiple sclerosis andthyroiditis. The peptide used in such treatment is an autoantigenpeptide. Thus, for example, for IDDM the peptide is the above-mentionedp277 peptide or the valine substituted analog p277(Val⁶-Val¹¹); formultiple sclerosis such peptide is derived from myelin basic protein;for thyroiditis the peptide is thought to be derived from thyroglobulin,and for rheumatoid arthritis the autoantigen can derive fromMycobacterium organisms, e.g., Mycobacterium tuberculosis.

[0029] It is not critical that the antigen be a peptide. Thus, forexample, TH1-mediated allergic responses which result in skinsensitivity and inflammation, such as contact dermatitis, can be treatedby a vaccine containing the irritant antigen and a biologically activecarrier in accordance with the present invention which will cause ashift in the cytokine response from a TH1-type to a TH2-type. Thus,while the patient will continue to have elevated antibody levels againstthe antigen, the inflammatory T cell response causing the skinirritation will be suppressed.

[0030] Accordingly, the tolerogenic biologically active carrier of thepresent invention may be used any time that it is desired to createtolerance for the antigen which the T cells are attacking, i.e., anytime that a vaccine is being used to restrict a T cell mediatedcondition, particularly a TH1 cell mediated condition. If it can bedetermined which antigen is activating the response in graft rejectionor in graft-versus-host disease, then the administration of such anantigen with a carrier in accordance with the present invention would beexpected to facilitate the shift of the undesirable inflammatory TH1response to a more desirable TH2 response, regardless of the overallcomplexity of the number of antigens to which T cells are active in suchcondition.

[0031] To determine the T-cell secretion of cytokines followingactivation with peptides, lymphocytes from the peripheral blood ofpatients are tested in an in vitro activation assay. Peripheral bloodlymphocytes are isolated from whole heparinized blood on ficol-hypaque,and cultured with the test peptide(s) at concentrations of 5-50 μg/ml.The supernatants from the cultured T-cells are collected at differenttime points and tested for activity of various cytokines, by ELISA orbioassay(s).

[0032] Examples of fat emulsions that can be used in the preparations ofthe present invention include, but are not limited to, the commerciallyavailable Intralipid and Lipofundin for intravenous nutrition, and thefat emulsions described in the above-mentioned U.S. Pat. Nos. 3,169,096,4,073,943 and 4,168,308, herein incorporated by reference in theirentirety. However, the finding according to the present invention thatthese metabolizable lipids, administered previously for intravenousnutrition, may be used effectively as vehicles for therapy of T cellmediated diseases, is completely unexpected. Similarly, the discoverythat these preparations are tolerogenic biologically active carrierswhich mediate a TH1→TH2 shift is also totally unexpected.

[0033] The fat emulsions of the present invention are preferably used asfreshly prepared or after storage in a container which is not open tothe atmospheric air. Prolonged storage of Intralipid, for example, whileexposed to atmospheric air, causes a decrease in the pH and acorresponding decrease in the biological activity.

[0034] In one embodiment, the biologically active carrier of theinvention is a fat emulsion comprising 10% soybean oil, 1.2% egg-yolkphospholipids, 2.5% glycerol and sterile water to complete 100 ml(Intralipid 10%). In another embodiment, the vehicle is a fat emulsioncomprising 20% soybean oil, 2.4% egg-yolk phospholipids, 2.5% glyceroland sterile water to complete 100 ml.

[0035] In yet another embodiment, the vehicle is a fat emulsioncomprising 5% soybean oil and another 5% triglycerides from animalorigin, e.g. 5% medium chain triglycerides from butter, 1.2% egg-yolklecithin, 2.5% glycerol and distilled water to complete 100 ml(Lipofundin 10%).

[0036] In one embodiment of the invention, the vehicle is a processedlipid emulsion obtained by centrifugation, e.g. at 10,000 g or higher,of the original fat emulsion defined herein, whereby a smalltriglyceride-rich (about 90% triglycerides) is formed on the top of aphospholipid-enriched aqueous dispersion containing about 1:1triglycerides:phospholipids. The two phases are separated and thephospholipid-rich aqueous dispersion is used as the vehicle.

[0037] The preparations of the invention may comprise one or morepeptides. Thus, for example, for the treatment of IDDM, the preparationmay comprise one or more of the peptides p12, p32, p277, p277(Val⁶),p277(Val¹¹), p277(Val⁶ -Val¹¹), or any of the other peptides of Table 1.In one preferred embodiment, the preparation for the treatment of IDDMcomprises a peptide p277 or p277(Val⁶-Val¹¹) and a fat emulsioncomprising 10% soybean oil, 1.2% egg-yolk phospholipids, 2.5% glyceroland sterile water to complete 100 ml (Intralipid 10%).

[0038] The invention further relates to the use of a fat emulsion asdefined herein or of a processed phospholipid-enriched aqueousdispersion prepared therefrom by centrifugation for the preparation of atherapeutic preparation comprising one or more peptides or otherantigens and said fat emulsion or processed aqueous dispersion as avehicle in the therapy of autoimmune diseases or other TH1 mediateddiseases or conditions.

[0039] The invention will now be illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

[0040] Peptide Therapy of Type I Diabetes Using p277(Val⁶-Val¹¹) in Oils

[0041] The efficacy of various lipid preparations as vehicles forpeptide therapy of the diabetes of NOD mice was tested. In this model,autoimmune destruction of the insulin producing β-cells in the pancreasis mediated by T-lymphocytes. An inflammatory infiltrate develops aroundthe pancreatic islets at 5-8 weeks of age and β-cell destruction leadingto insulin deficiency and overt diabetes becomes manifested at 14-20weeks of age affecting almost 100% of female NOD mice by 35-40 weeks ofage.

[0042] NOD female mice were treated with 100 μg of peptidep277(Val⁶-Val¹¹) per mouse sc in 0.1 ml of: (i) Phosphate-bufferedsaline (PBS), or (ii) a 10% lipid emulsion composed of 10% soybean oil,1.2% egg phospholipids and 2.25% glycerol (Intralipid, Kabi PharmaciaAB, Sweden).

[0043] The incidence of diabetes at 6 months of age and the productionof anti-p277(Val⁶-Val¹¹) antibodies was followed. Diabetes was diagnosedas persistent hyperglycemia, blood glucose levels over 11 mmol/Lmeasured at least twice at weekly intervals with a Beckman GlucoseAnalyzer II. Successful peptide treatment was assayed by maintenance ofa normal blood glucose concentration (less than 11 mmol/L), remission ofthe intra-islet inflammation of the pancreatic islets (insulitis) andinduction of antibodies to the therapeutic peptide as an indicator of aTH2-type immune response. The results are shown in Table 2. TABLE 2Incidence of Diabetes at 6 months. Treatment Death (%) (%) Diabetesincidence p277 (Val⁶-Val¹¹)/PBS  90 80 p277 (Val⁶-Val¹¹)/Intralipid  45# 20# none 100 90

[0044] As can be seen from Table 2, peptide treatment administered inIntralipid was effective in reducing the incidence of diabetes anddeath. On the other hand, treatment administered in PBS was ineffective.

Example 2

[0045] Anti-p277(Val⁶-Val¹¹) Antibody Production

[0046] The protection from diabetes by treatment with thep277(Val⁶-Val¹¹) peptide is dependent on TH2 immunological reactivity tothe peptide. Therefore, antibody production was measured in thep277(Val⁶-Val¹¹)-immunized mice by ELISA. Maxisorp microtiter plates(Nunc) were coated with p277(Val⁶-Val¹¹) peptide, 10 μg/ml, for 18 h andnon-specific binding blocked with 7% milk powder for 2 h. The mousesera, diluted 1:50, were allowed to bind for 2 h and the specificbinding was detected by adding alkaline phosphatase anti-mouse IgG(Serotec) for 2 h and p-nitrophenylphosphate substrate (Sigma) for 30min. The color intensity was measured by an ELISA reader (Anthos) atOD=405 nm.

[0047] As can be seen from FIG. 1, NOD mice immunized top277(Val⁶-Val¹¹) in Intralipid developed peptide specific antibodies,while mice immunized to p277(Val⁶-Val¹¹) in PBS showed no antibodyresponses at all.

Example 3

[0048] Antibody Isotypes Induced by p277(Val⁶-Val¹¹) Therapy

[0049] The association of p277(Val⁶-Val¹¹) Intralipid therapy withantibodies to p277(Val⁶-Val¹¹) shown in Example 2, suggested that thetherapeutic effect might result from a shift in the predominantcytokines produced by the autoimmune T cells. T cells of the CD4“helper” type have been divided into two groups by the characteristiccytokines they secrete when activated (Mosmann and Coffman, 1989): TH1cells secrete IL-2, which induces T-cell proliferation, and cytokinessuch as IFNγ, which mediate tissue inflammation; TH2 cells, in contrast,secrete IL-4, which “helps” B cells produce certain antibody isotypes,and IL-10 and other cytokines, which can “depress” tissue inflammation.The possibility of a shift from TH1 to TH2-like behavior was supportedby analysis of the isotypes of the antibodies produced afterp277(Val⁶-Val¹¹) therapy.

[0050] Groups of NOD mice, 3 months old, were treated withp277(Val⁶-Val¹¹) or with PBS in oil as described in Example 2. The seraof individual mice were assayed for the isotypes of their antibodies top277(Val6-Val¹¹) after treatment (12-15 mice per group). The antibodyisotypes were detected using an ELISA assay with isotype-specificdeveloping antibody reagents (Southern Biotechnology Associates,Birmingham, Ala.). The results are shown in FIG. 2, wherein: Antibodiesto p277(Val⁶-Val¹¹) in control-treated NOD mice—open circles; inp277(Val⁶-Val¹¹)-treated mice—closed circles. The columns in eachexperiment show results from equal numbers of mice; an apparentreduction in numbers of circles is caused by superimposition.

[0051] Analysis of the antibody isotypes of the anti-p277 antibodiesdeveloping after treatment showed them to be exclusively of the IgG1 andIgG2b classes, dependent on TH2 T cells producing IL-4 (Snapper et al.,1993a) and possibly TGFβ (Snapper et al., 1993b). There were no TH1-typeIgG2a antibodies induced by p277(Val⁶-Val¹¹) therapy. The development ofantibodies to the specific peptide used in treatment is a sign that theautoimmune T-cell responses have shifted from a damaging inflammatorymode called TH1 to a TH2 T-cell response that produces innocuousantibodies and suppresses inflammation and tissue damage (Rabinovitch,1994).

Example 4

[0052] Peptide p277(Val⁶-Val¹¹)/Intralipid Therapy Induces a SpecificSwitch in the Cytokine Profile

[0053] To confirm the idea of a cytokine switch, the cytokines producedby the T cells reactive to the p277(Val⁶-Val¹¹) in thep277(Val⁶-Val¹¹)/Intralipid-treated and control mice were assayed.Concanavalin A (ConA), a T-cell mitogen, was used to activate totalsplenic T-cells as a control.

[0054] Groups of 10 NOD mice, 3 months old, were treated withp277(Val⁶-Val¹¹) in Intralipid (closed bars) or with PBS in Intralipid(open bars; see Example 2). Five weeks later, the spleens of the micewere removed and the spleen cells were pooled. The spleen cells wereincubated with Con A or p277(Val⁶-Val¹¹) for 24 h (for IL-2 and IL-4secretion) or for 48 h (for IL-10 and IFNγ secretion). The presence ofthe cytokines in the culture supernatants was quantitated by ELISA,using Pharmingen paired antibodies according to the Pharmingen cytokineELISA protocol. Pharmingen recombinant mouse cytokines were used asstandards for calibration curves. Briefly, flat-bottom 96-wellmicrotiter plates were coated with rat anti-mouse cytokine mAbs for 18 hat 4° C., and the culture supernatants or recombinant mouse cytokineswere added for 18 h at 4° C. The plates were washed, and biotinylatedrat anti-mouse cytokine mAbs were added for 45 min at room temperature,then extensively washed, and avidin-alkaline phosphatase was added. Theplates were washed, a chromogen substrate (p-nitrophenylphosphate) wasadded and samples were read at 405 nm in an ELISA reader. The resultsare shown in FIG. 3. The concentrations of cytokines are shown as the ODreadings. *P<0.01.

[0055]FIG. 3A shows that the spleen cells of control mice secreted bothIL-2 and IFNγ upon incubation with p277(Val⁶-Val¹¹). In contrast, thep277(Val⁶-Val¹¹)-treated mice produced significantly less (P<0.01) IL-2and IFNγ in response to incubation with peptide p277(Val⁶-Val¹¹). Thisreduction in TH1 cytokines was specific; the p277(Val⁶-Val¹¹)-treatedmice maintained their IL-2 and IFNγ cytokine responses to ConA (FIG.3B). FIGS. 3A and 3B show the amounts of IL-10 and IL-4 produced by thespleen cells of the mice. The control mice produced very little IL-4 orIL-10 in response to p277(Val⁶-Val¹¹) or Con A. In contrast, there was asignificant increase in IL-10 and IL-4 in response only top277(Val⁶-Val¹¹) and only in the p277(Val⁶-Val¹¹)/Intralipid-treatedmice (P<0.01). A decrease in IL-2 and IFNγ coupled with an increase inIL-10 and IL-4 confirms the shift from TH1-like behavior to TH2-likebehavior. Such a shift might help explain both a decline in T-cellproliferation to p277 shown previously by the inventors (Elias et al.,1991) and the appearance of IgG1 and IgG2b antibodies top277(Val⁶-Val¹¹) according to the present invention.

Example 5

[0056] Spontaneous T-Cell Proliferative Responses to p277(Val⁶-Val¹¹) isReduced by p277(Val⁶-Val¹¹) Therapy

[0057] Groups of 5 female mice of the NOD/Lt strain were treated at theage of 3 months with 100 μg of peptide p277(Val⁶-Val¹¹) in Intralipid orwith PBS mixed with Intralipid, sc in the back. Five weeks later, thespleens of the mice were removed and the T-cell proliferative responseswere assayed in vitro to the T-cell mitogen Con A (1.25 μg/ml) or top277(Val⁶-Val¹¹) (10 μg/ml) using a standard assay. The results areshown in FIG. 4, wherein: Con A-black striped bars;p277(Val⁶-Val¹¹)—grey bars. The T-cell responses were detected by theincorporation of [³H] thymidine added to the wells in quadruplicatecultures for the last 18 hours of a 3-day culture. The stimulation index(SI) was computed as the ratio of the mean cpm of test cultures to themean cpm of antigen-containing wells to control wells cultured withoutantigens or Con A. The standard deviations from the mean cpm were alwaysless than 10%.

[0058] As shown in FIG. 4, the control mice tested with PBS/Intralipidshowed T-cell proliferative responses to both p277(Val⁶-Val¹¹) and tothe T-cell mitogen Con A. In contrast, the mice treated withp277(Val⁶-Val¹¹) in Intralipid showed a decrease in T-cell proliferativereactivity to p277(Val⁶-Val¹¹) but no decrease to Con A. Thus thebeneficial effect of p277(Val⁶-Val¹¹) peptide therapy is caused not byinactivating the autoimmune response, but by activating the autoimmunityinto a different cytokine mode of behavior (Cohen, 1995). Regulation ofdestructive autoimmunity is programmed within the immune system (Cohen,1992); it need only be activated by a suitable signal which requires thepeptide together with the lipid vehicle; neither the peptide alone orthe lipid without the peptide are effective, as shown in Table 1. Theseresults indicate that metabolizable lipid emulsions may be usedefectively as vehicles for therapy of autoimmune diseases. Each diseasewill require its own specific peptide, but the metabolizable lipidemulsion can be used for the various therapies.

Example 6

[0059] Administration of Peptide in Intralipid Affects Development ofExperimental Autoimmune Encephalomyelitis

[0060] Experimental autoimmune encephalomyelitis (EAE) is anexperimental autoimmune disease of animals that is thought to modelaspects of multiple sclerosis (Zamvil and Steinman, 1990). EAE can beinduced in susceptible strains of rats, such as the Lewis rat, byimmunization to myelin basic protein (MBP) in complete Freund's adjuvant(CFA), an emulsion of mineral oil containing killed Mycobacteria. Thedisease develops about 12 days after immunization and is characterizedby paralysis of various degrees due to inflammation of the centralnervous system. The paralysis can last up to 6 or 7 days and the ratsusually recover unless they die during the peak of their acuteparalysis. EAE is caused by T cells that recognize defined determinantsof the MBP molecule. The major MBP determinant in the Lewis rat iscomposed of the peptide sequence 71-90 (Zamvil and Steinman, 1990).

[0061] We therefore performed an experiment to test whetheradministration of the encephalitogenic MBP peptide p71-90 in IFA couldalso inhibit the development of EAE. FIG. 5 shows that theadministration of p71-90 in IFA 14 days before the induction of EAE ledto a significant decrease in the maximal degree of paralysis compared tothe control treatment with PBS emulsified in IFA, which had no effect onthe severity of the disease. Thus, p71-90 given in IFA affects EAE.

[0062] However, IFA cannot be administered, as stated above, to humansbecause it is not metabolizable in the body and causes localinflammation. We therefore treated Lewis rats with p71-90 in Intralipid.FIG. 6 shows the results. The rats that had received p71-90 inIntralipid developed significantly less paralysis than did the controlrats treated with PBS/Intralipid. Therefore, it can be concluded that arelevant peptide such as p71-90 administered in Intralipid is capable ofmodulating EAE in rats. Hence, the effects of peptide/Intralipidtreatment are not limited to only one peptide, in one species, or toonly one autoimmune disease.

Example 7

[0063] Effectiveness of New vs. Aged 10% Intralipid Emulsion

[0064] 10% Intralipid emulsion was used to treat 12 week old NOD femalemice with p277(Val⁶-Val¹¹). The emulsion was used either on the day thesealed bottle was opened, or 4 months later, after exposure toatmospheric air. The pH of the emulsion was tested at the time ofpreparing the peptide+emulsion for treatment. Aging was marked by a fallin pH from 8.2 to 6.7. In each experiment 10 mice were treated with thepeptide+emulsion preparation, 10 mice received the emulsion alone, and10 mice were untreated. The results are shown in Table 3. TABLE 3Emulsion Diabetes Mortality Group Treatment pH (%) (%) 1 peptide +emulsion 8.2  20*  10* 2 emulsion ″ 90 70 3 peptide + emulsion 6.7 60 404 emulsion ″ 80 60 5 untreated — 90 80

[0065] It can be seen that the placebo-treated mice (emulsion only,groups 2 and 4) and the untreated mice (group 5) developed a similarincidence of diabetes, 80-90% at 6 months of age. In contrast, treatingthe mice with peptide in the newly opened emulsion protected 80% of themice from diabetes. However, using the “aged” emulsion only protected40%. Therefore, the emulsion was chemically unstable after exposure toair, as shown by the marked decrease in pH value. This change isrelevant to its biological activity. Hence, the Intralipid is abiologically active carrier whose functional properties depend on the pHand not only on the presence of inert lipid.

[0066] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

[0067] While this invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications. This application is intended to cover anyvariations, uses, or adaptations of the inventions following, ingeneral, the principles of the invention and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the invention pertains and as may be applied tothe essential features hereinbefore set forth as follows in the scope ofthe appended claims.

[0068] All references cited herein, including journal articles orabstracts, published or unpublished U.S. or foreign patent applications,issued U.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

[0069] Reference to known method steps, conventional methods steps,known methods or conventional methods is not in any way an admissionthat any aspect, description or embodiment of the present invention isdisclosed, taught or suggested in the relevant art.

[0070] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying knowledge within the skill of the art (including the contentsof the references cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present application.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology of the present specification is to be interpretedby the skilled artisan in light of the teachings and guidance presentedherein, in combination with the knowledge of one of ordinary skill inthe art.

1 4 573 amino acids amino acid single linear protein 1 Met Leu Arg LeuPro Thr Val Phe Arg Gln Met Arg Pro Val Ser Arg 1 5 10 15 Val Leu AlaPro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30 Gly Ala AspAla Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu Ala 35 40 45 Asp Ala ValAla Val Thr Met Gly Pro Lys Gly Arg Thr Val Ile Ile 50 55 60 Glu Gln GlyTrp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val 65 70 75 80 Ala LysSer Ile Asp Leu Lys Asp Lys Tyr Lys Asn Ile Gly Ala Lys 85 90 95 Leu ValGln Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110 ThrThr Thr Ala Thr Val Leu Ala Arg Ser Ile Ala Lys Glu Gly Phe 115 120 125Glu Lys Ile Ser Lys Gly Ala Asn Pro Val Glu Ile Arg Arg Gly Val 130 135140 Met Leu Ala Val Asp Ala Val Ile Ala Glu Leu Lys Lys Gln Ser Lys 145150 155 160 Pro Val Thr Thr Pro Glu Glu Ile Ala Gln Val Ala Thr Ile SerAla 165 170 175 Asn Gly Asp Lys Glu Ile Gly Asn Ile Ile Ser Asp Ala MetLys Lys 180 185 190 Val Gly Arg Lys Gly Val Ile Thr Val Lys Asp Gly LysThr Leu Asn 195 200 205 Asp Glu Leu Glu Ile Ile Glu Gly Met Lys Phe AspArg Gly Tyr Ile 210 215 220 Ser Pro Tyr Phe Ile Asn Thr Ser Lys Gly GlnLys Cys Glu Phe Gln 225 230 235 240 Asp Ala Tyr Val Leu Leu Ser Glu LysLys Ile Ser Ser Ile Gln Ser 245 250 255 Ile Val Pro Ala Leu Glu Ile AlaAsn Ala His Arg Lys Pro Leu Val 260 265 270 Ile Ile Ala Glu Asp Val AspGly Glu Ala Leu Ser Thr Leu Val Leu 275 280 285 Asn Arg Leu Lys Val GlyLeu Gln Val Val Ala Val Lys Ala Pro Gly 290 295 300 Phe Gly Asp Asn ArgLys Asn Gln Leu Lys Asp Met Ala Ile Ala Thr 305 310 315 320 Gly Gly AlaVal Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335 Val GlnPro His Asp Leu Gly Lys Val Gly Glu Val Ile Val Thr Lys 340 345 350 AspAsp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gln Ile Glu 355 360 365Lys Arg Ile Gln Glu Ile Ile Glu Gln Leu Asp Val Thr Thr Ser Glu 370 375380 Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly 385390 395 400 Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val AsnGlu 405 410 415 Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg AlaAla Val 420 425 430 Glu Glu Gly Ile Val Leu Gly Gly Gly Cys Ala Leu LeuArg Cys Ile 435 440 445 Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu AspGln Lys Ile Gly 450 455 460 Ile Glu Ile Ile Lys Arg Thr Leu Lys Ile ProAla Met Thr Ile Ala 465 470 475 480 Lys Asn Ala Gly Val Glu Gly Ser LeuIle Val Glu Lys Ile Met Gln 485 490 495 Ser Ser Ser Glu Val Gly Tyr AspAla Met Ala Gly Asp Phe Val Asn 500 505 510 Met Val Glu Lys Gly Ile IleAsp Pro Thr Lys Val Val Arg Thr Ala 515 520 525 Leu Leu Asp Ala Ala GlyVal Ala Ser Leu Leu Thr Thr Ala Glu Val 530 535 540 Val Val Thr Glu IlePro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala 545 550 555 560 Met Gly GlyMet Gly Gly Gly Met Gly Gly Gly Met Phe 565 570 24 amino acids aminoacid single linear peptide 2 Val Leu Gly Gly Gly Val Ala Leu Leu Arg CysIle Pro Ala Leu Asp 1 5 10 15 Ser Leu Thr Pro Ala Asn Glu Asp 20 24amino acids amino acid single linear peptide 3 Val Leu Gly Gly Gly CysAla Leu Leu Arg Val Ile Pro Ala Leu Asp 1 5 10 15 Ser Leu Thr Pro AlaAsn Glu Asp 20 24 amino acids amino acid single linear peptide 4 Val LeuGly Gly Gly Val Ala Leu Leu Arg Val Ile Pro Ala Leu Asp 1 5 10 15 SerLeu Thr Pro Ala Asn Glu Asp 20

1. A therapeutic preparation for treatment of a T cell mediated diseaseor condition comprising an antigen and a biologically active carrier,wherein the antigen is an antigen recognized by inflammatory T cellsassociated with the pathogenesis of said disease or condition, andwherein the said carrier is a fat emulsion comprising 10-20%triglycerides of plant and/or animal origin, 1.2-2.4% phospholipids ofplant and/or animal origin, 2.25-4.5% osmo-regulator, 0-0.05%anti-oxidant, and sterile water to complete 100 ml.
 2. A preparationaccording to claim 1, wherein the triglycerides are of plant origin. 3.A preparation according to claim 2, wherein the triglycerides arederived from soybean oil.
 4. A preparation according to claim 1, whereinthe triglycerides are of animal origin.
 5. A preparation according toclaim 4, wherein the triglycerides are derived from egg yolk or bovineserum.
 6. A preparation according to any one of claims 1 to 5, whereinthe phospholipids are of plant origin.
 7. A preparation according toclaim 6, wherein the phospholipids are derived from soybeans.
 8. Apreparation according to any one of claims 1 to 5, wherein thephospholipids are of animal origin.
 9. A preparation according to claim8, wherein the phospholipids are derived from egg yolk or bovine serum10. A preparation according to any one of claims 1 to 5, wherein theosmo-regulator is selected from the group comprising glycerol, sorbitoland xylitol.
 11. A preparation according to any one of claims 1 to 5,comprising 0.05% tocopherol as anti-oxidant.
 12. A preparation accordingto claim 1, wherein the biologically active carrier is a fat emulsioncomprising 10% soybean oil, 1.2% egg-yolk phospholipids, 2.5% glyceroland sterile water to complete 100 ml.
 13. A preparation according toclaim 1, wherein the biologically active carrier is a fat emulsioncomprising 20% soybean oil, 2.4% egg-yolk phospholipids, 2.5% glyceroland sterile water to complete 100 ml.
 14. A preparation according toclaim 1, wherein the biologically active carrier is a fat emulsioncomprising 5% soybean oil, 5% medium chain triglycerides, 1.2% egg-yolklecithin, 2.5% glycerol and sterile water to complete 100 ml.
 15. Apreparation according to any one of claims 1 to 5 or 12 to 14 whichcauses shifting of an individual's T-cell cytokine response from TH1 toTH2.
 16. A preparation according to any one of claims 1 to 5 or 12 to 14which causes a decrease in IL-2 or IFN-γ T-cell cytokine response and anincrease in IL-4 or IL-10 T-cell cytokine response.
 17. A preparationaccording to any one of claims 1 to 5 or 12 to 14 for the treatment ofinsulin dependent diabetes mellitus (IDDM) comprising a peptide derivedfrom the human heat shock protein 60 (hsp60) that is recognized byinflammatory T-cells associated with the pathogenesis of IDDM whereinsaid peptide is selected from the group of peptides listed in Table 1.18. A preparation according to claim 17 for the treatment of IDDMcomprising the peptide p277 and of a fat emulsion comprising 10% soybeanoil, 1.2% egg-yolk phospholipids, 2.5% glycerol and sterile water tocomplete 100 ml.
 19. A preparation according to claim 17 for thetreatment of IDDM comprising the peptide p277(Val⁶-Val¹¹) and a carrierconsisting of a fat emulsion comprising 10% soybean oil, 1.2% egg-yolkphospholipids, 2.5% glycerol and sterile water to complete 100 ml.
 20. Apreparation according to any one of claims 1 to 5 or 12 to 14 for thetreatment of multiple sclerosis comprising a peptide derived from myelinbasic protein (MBP) that is recognized by T-cells involved in thepathogenesis of multiple sclerosis.
 21. Use of a fat emulsion comprising10-20% triglycerides of plant and/or animal origin, 1.2-2.4%phospholipids of plant and/or animal origin, 2.25-4.5% osmo-regulator,0-0.05% anti-oxidant, and sterile water to complete 100 ml for themanufacture of a therapeutic preparation according to claim
 1. 22. Useof a fat emulsion comprising 10% soybean oil, 1.2% egg-yolkphospholipids, 2.5% glycerol, and sterile water to complete 100 ml, forthe manufacture of a therapeutic preparation according to claim
 12. 23.A method of treatment of a patient suffering from a T cell mediateddisease or condition which comprises administering to said patient apreparation comprising an antigen recognized by inflammatory T cellsassociated with the pathogenesis of said disease or condition in abiologically active carrier consisting of a fat emulsion comprising10-20% triglycerides of plant and/or animal origin, 1.2-2.4%phospholipids of plant and/or animal origin, 2.25-4.5% osmo-regulator,0-0.05% anti-oxidant, and sterile water to complete 100 ml.
 24. A methodaccording to claim 23, wherein the carrier consists of fat emulsioncomprising 10% soybean oil, 1.2% egg-yolk phospholipids, 2.5% glycerol,and sterile water to complete 100 ml.
 25. A method in accordance withclaim 23 or claim 24, wherein said T cell mediated disease is anautoimmune disease and said antigen is a peptide.
 26. A method inaccordance with claim 23 or claim 24, wherein said T cell mediateddisease is a TH1 mediated disease.
 27. A method in accordance with claim26, wherein said autoimmune disease is an organ-specific autoimmunedisease.
 28. A method of treatment of a patient suffering from IDDMwhich comprises administering to said patient a preparation comprisingone or more peptides listed in Table 1 and a biologically active carrierconsisting of a fat emulsion comprising 10% soybean oil, 1.2% egg-yolkphospholipids, 2.5% glycerol, and sterile water to complete 100 ml. 29.A method according to claim 28, wherein the preparation comprises thepeptide p277.
 30. A method according to claim 28, wherein thepreparation comprises the peptide p277(Val⁶-Val¹¹).
 31. A method inaccordance with claim 23 or claim 24, wherein said T cell mediateddisease is a T cell mediated allergic condition and said antigen is theallergen which triggered said condition.